REVIEW ARTICLE
How Saccharomyces cerevisiae copes with toxic metals and
metalloids
Robert Wysocki1 & Markus J. Tamás2
1
Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland; and 2Department of Cell and Molecular Biology/Microbiology,
University of Gothenburg, Gothenburg, Sweden
Correspondence: Markus J. Tamás,
Department of Cell and Molecular Biology/
Microbiology, University of Gothenburg, PO
Box 462, S-405 30 Gothenburg, Sweden. Tel.:
146 31 786 2548; fax: 146 31 786 2599;
e-mail: [email protected]
Received 5 February 2010; accepted 28
February 2010.
Final version published online 31 March 2010.
DOI:10.1111/j.1574-6976.2010.00217.x
Abstract
Toxic metals and metalloids are widespread in nature and can locally reach fairly
high concentrations. To ensure cellular protection and survival in such environments, all organisms possess systems to evade toxicity and acquire tolerance. This
review provides an overview of the molecular mechanisms that contribute to metal
toxicity, detoxification and tolerance acquisition in budding yeast Saccharomyces
cerevisiae. We mainly focus on the metals/metalloids arsenic, cadmium, antimony,
mercury, chromium and selenium, and emphasize recent findings on sensing and
signalling mechanisms and on the regulation of tolerance and detoxification
systems that safeguard cellular and genetic integrity.
Editor: Martin Kupiec
MICROBIOLOGY REVIEWS
Keywords
arsenic; cadmium; chromium; selenium; metal
toxicity; metal detoxification.
Introduction
Metals and metalloids profoundly affect biological systems
either because they are essential or because they are toxic or
harmful when present in excessive amounts. Living organisms have always dealt with metals, and a very large number
of proteins have evolved that require metals for catalytic
activity and/or for maintaining protein structure (Waldron
et al., 2009). Cells also utilize a wide array of homeostasis
and tolerance mechanisms that regulate the availability of
essential metals and limit the damaging effects of toxic
elements. Malfunction of metal homeostatic or detoxification systems may cause a range of human diseases including
cancer. At the same time, many metals are also used as
therapeutic agents to treat various ailments and disorders
(Thompson & Orvig, 2003; Tamás & Martinoia, 2005;
Beyersmann & Hartwig, 2008). The importance of metals
for human health as well as their impact on the environment
has spurred research on metal biology and led to a significant progress in understanding metal responses and tolerance acquisition mechanisms in many prokaryotic and
eukaryotic organisms. Despite this, relatively little is known
about toxicity mechanisms at a molecular level. Likewise,
FEMS Microbiol Rev 34 (2010) 925–951
our knowledge of metal sensing and how this information is
translated into appropriate cellular responses is scarce.
Saccharomyces cerevisiae (budding yeast) has proved to be a
powerful model organism to unravel the molecular details of
metal action and detoxification strategies. This review highlights various aspects of metal toxicity and tolerance in
S. cerevisiae, with a special emphasis on the sensing, signalling and regulatory mechanisms used in response to nonessential metals and metalloids including arsenic, cadmium,
antimony, mercury, chromium and selenium.
Metal-induced damage and toxicity
The impact of metals and metalloids on biological systems
ranges from essential, through beneficial, to nonessential
and highly toxic. The toxicity of a given metal is governed by
its mechanisms of uptake, oxidation state and speciation,
intracellular distribution and interactions with various
macromolecules, and depends on its physicochemical properties and ligand preferences. ‘Soft’ transition metals such as
silver (Ag), cadmium (Cd) and mercury (Hg) have sulphur
as their preferred ligand. ‘Hard’ transition metals such as
chromium (Cr), manganese (Mn) and molybdenum (Mo),
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and the metalloids arsenic (As), antimony (Sb), selenium
(Se), tellurium (Te) and bismuth (Bi) prefer oxygen in their
higher oxidation states, while they prefer sulphur in their
lower oxidation states. Lead (Pb), iron (Fe), cobalt (Co),
nickel (Ni), copper (Cu) and zinc (Zn) can use oxygen,
sulphur or nitrogen as ligands (Summers, 2009). Hence,
nearly all nonessential metals and metalloids, including Hg,
As, Cd and Pb, display high reactivity with sulphhydryl
groups. This property may contribute to their toxicity, but is
also exploited by cells for detoxification. Several compounds
containing these metals are classified as human carcinogens
(e.g. As, Cd, Cr, Ni) or probable carcinogens (e.g. Pb, Co)
according to the International Agency for Research on
Cancer (http://www.iarc.fr). Metal toxicity may be caused
by oxidative stress, impaired DNA repair, inhibition of
enzyme function and by disturbing the function of proteins
that regulate proliferation, cell cycle progression, apoptosis
or differentiation (Stohs & Bagchi, 1995; Ercal et al., 2001;
Chen & Shi, 2002; Harris & Shi, 2003; Beyersmann &
Hartwig, 2008) (Fig. 1).
Metals and oxidative stress
Oxidative stress originates from toxic levels of oxygenderived reactive species. Reactive oxygen species (ROS) are
mainly singlet oxygen (O), superoxide anion (O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH). ROS can
Fig. 1. Metal toxicity mechanisms and metal responses. Many metals
trigger oxidative stress in cells, interfere with protein function and activity
and/or impair DNA repair mechanisms either directly or indirectly. In
response to metal exposure, cells arrest cell cycle progression, alter gene
expression and metabolism, and adjust transport processes to safeguard
cellular and genetic integrity.
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R. Wysocki & M.J. Tamás
attack and damage all cellular macromolecules, leading to
protein oxidation, lipid peroxidation and DNA damage
(Halliwell & Gutteridge, 1984). The mechanisms leading to
metal-induced oxidative stress in vivo are largely elusive.
While Fe, Cu, Cr and Co can undergo redox-cycling reactions, Cd, Hg and Pb are redox inactive. Nevertheless, the
latter metals may induce oxidative stress by inhibiting
specific enzymes, by depleting pools of antioxidants or
through other indirect mechanisms (Stohs & Bagchi, 1995;
Ercal et al., 2001; Beyersmann & Hartwig, 2008). Cd has
been shown to induce oxidative stress and lipid peroxidation
in yeast (Brennan & Schiestl, 1996; Howlett & Avery, 1997).
Arsenite [As(III)] – the most toxic form of As – triggers
increased ROS production in mammals (Liu et al., 2001),
but not to any large extent in wild-type yeast (Menezes et al.,
2008). Nevertheless, As(III)-induced oxidative stress and
lipid peroxidation were detected in mutants with impaired
As(III) detoxification (yap8D cells) or oxidative stress defence (yap1D cells) systems (Menezes et al., 2008), indicating
that As(III) enhances ROS levels also in yeast. While Cd and
As have no biological functions, selenium [Se(0)] is an
essential trace element and nontoxic at low levels. However,
selenate [Se(VI)], selenite [Se(III)] and selenide [Se(II)] are
highly reactive and may cause increased ROS production.
Indeed, certain reactions of the pathway involved in the
reduction of Se(III) into Se(0) appear to produce O2 and
H2O2 (Seko & Imura, 1997; Turner et al., 1998). In a similar
way, reduction of chromate [Cr(VI)] – the most toxic form
of Cr – into the less toxic form chromite [Cr(III)] produces
ROS. In this case, it is the reduction intermediates Cr(V)
and Cr(IV) that are thought to trigger OH generation
through a Fenton-like mechanism (Shi et al., 1994; Stohs &
Bagchi, 1995; Beyersmann & Hartwig, 2008). Fenton-type
reactions are described for the nutrient metals Fe and Cu
and are supposed to be a major source of hydroxyl radicals
and oxidative stress in the cell (Halliwell & Gutteridge,
1984). As mentioned above, several metals are unable to
undergo such reactions. Nevertheless, redox-inactive metals
may perturb intracellular Fe metabolism (Kitchin & Wallace, 2008), leading to increased levels of free Fe in the cell,
which in turn could enhance Fenton-type reactions and
elevated ROS levels.
An often-cited mechanism of metal toxicity is depletion of
glutathione (GSH) (e.g. Stohs & Bagchi, 1995), which is the
main antioxidant molecule in cells. Saccharomyces cerevisiae
neutralizes many metals and metalloids, for example Cd,
As(III), Hg and antimonite [Sb(III)] through chelation to
GSH (see later in text); hence, it is possible that this process
leads to reduced cytosolic GSH levels. GSH depletion would
influence the redox environment and impair the activities of
GSH-dependent enzymes, such as glutathione peroxidases,
glutathione S-transferases and glutaredoxins, thereby affecting many cellular processes. However, metal concentrations
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Yeast metal tolerance
that are high enough to be toxic may still be too low to
significantly deplete cytosolic GSH. For instance, the GSH
concentration is in the millimolar range in yeast, whereas Cd
is toxic in the micromolar range (Lafaye et al., 2005).
Another observation that argues against GSH depletion as a
general toxicity mechanism, at least in S. cerevisiae, is that
GSH levels in fact strongly increase in response to Cd (Lafaye
et al., 2005) and As(III) (Thorsen et al., 2007) exposure (see
later in text). Nevertheless, it cannot be excluded that some
metals reduce the GSH pool to an extent where GSHdependent enzyme activities are affected.
Impact on proteins
Metals and metalloids have the capacity to bind to proteins,
often via thiol groups of cysteine residues, and inhibit
enzyme function. For instance, methylmercury (MeHg)
strongly inhibits the yeast L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT), which catalyses the synthesis of glutamine-6-phosphate. Overexpression of GFAT
results in MeHg resistance, suggesting that GFAT is targeted
by MeHg (Naganuma et al., 2000). Whether MeHg inhibits
this enzyme by binding to a thiol group is unknown. Cd
inhibits human thiol transferases [glutathione reductase,
thioredoxin reductase (TrxR), thioredoxin] in vitro, possibly
by binding to vicinal cysteines in their active sites (Chrestensen et al., 2000). As these proteins protect cells from
oxidative stress, their inhibition would lead to increased
ROS levels. Cd may also displace Zn and calcium (Ca) ions
from metalloproteins (Stohs & Bagchi, 1995; Schützendübel
& Polle, 2002; Faller et al., 2005) and zinc finger proteins
(Hartwig, 2001), thereby affecting their activity, but it is not
known how much this mechanism contributes to Cd
toxicity. As(III) has been shown to interact with actin,
tubulin, TrxR and many other proteins (Hoffman & Lane,
1992; Menzel et al., 1999; Zhang et al., 2007; Kitchin &
Wallace, 2008), and the classical view is that binding leads to
enzyme inhibition. This concept is supported by the fact
that As(III) binds b-tubulin and inhibits tubulin polymerization (Zhang et al., 2007). In contrast, As(III) binding to
pyruvate kinase does not impair enzyme activity (Zhang
et al., 2007). Similarly, As(III) binds to the pyruvic acid
dehydrogenase (PDH) multienzyme complex, but PDH
appears to be more sensitive to inhibition by ROS than by
As-containing agents (Samikkannu et al., 2003). Hence,
certain proteins may be more susceptible to As(III)-induced
protein oxidation than to direct binding of As(III) to critical
thiols (Samikkannu et al., 2003). Arsenic trioxide, the form
of arsenite used in cancer therapy, has been shown to inhibit
mammalian TrxR, probably by direct binding to the enzyme.
TrxR inhibition leads to the oxidation of thioredoxin, which
is one of the main thiol-dependent electron donor systems
in mammalian cells, thereby affecting the regulation of the
FEMS Microbiol Rev 34 (2010) 925–951
cellular redox environment and a wide range of cellular
activities (Lu et al., 2007). Finally, Cr exposure has been
shown to trigger oxidative protein damage (Sumner et al.,
2005) and enhanced protein aggregation (Holland et al.,
2007) in yeast. Whether protein aggregation is a general
feature of metal-exposed cells is not known.
Inhibition of DNA repair
Most metals are weak mutagens and do not damage DNA
directly; instead, they may trigger genotoxicity by interfering
with DNA repair processes (Beyersmann & Hartwig, 2008).
Cd induces recombination events, and base-substitution
and frame-shift mutations (Brennan & Schiestl, 1996; Jin
et al., 2003; Serero et al., 2008) by inhibiting the DNA
mismatch repair system (Jin et al., 2003). This inhibition
is a result of Cd blocking the ATPase activity of the
Msh2p–Msh6p complex (Banerjee & Flores-Rozas, 2005),
but it is not known whether Cd binds to a specific site or
displaces a critical Zn ion (McMurray & Tainer, 2003;
Banerjee & Flores-Rozas, 2005). As(III) can also trigger
various types of DNA damage in mammalian systems and
it has been proposed that this metalloid interferes with DNA
repair systems (Shi et al., 2004). However, DNA repair
functions were not enriched among yeast genes sensitive to
As(III) (Haugen et al., 2004; Thorsen et al., 2009) and
As(III) treatment did not increase DNA double-strand
brakes in yeast (Jo et al., 2009). Se(III) is slightly mutagenic
for yeast and the error-prone repair pathway appears to be
important for tolerance; the rev3D mutant, which is defective in error-prone repair, is strongly Se(III) sensitive while
mutations in other DNA repair pathways do not affect
tolerance to any large extent (Pinson et al., 2000). How the
other metals described here affect DNA repair systems is not
known.
Other toxicity mechanisms
Besides the general toxicity mechanisms described above
(Fig. 1), other modes of metal action exist. For example, Cr
toxicity appears to involve mRNA mistranslation (Holland
et al., 2007); As(III) toxicity involves disruption of the actin
and tubulin cytoskeleton as well as the folding of de novo
synthesized actin and tubulin monomers (Thorsen et al.,
2009); Cd toxicity appears to target DNA replication (Serero
et al., 2008) and triggers Fe deficiency (Ruotolo et al., 2008;
Thorsen et al., 2009). Many metals such as Fe, Cu, Zn, Se, Cr,
Cd, Hg and Pb influence membrane fluidity, which in turn
could contribute to their toxicity (Garcia et al., 2005).
Finally, recent in vitro studies indicate that metals interfere
with proper protein folding (Sharma et al., 2008; Ramadan
et al., 2009). Even though the above studies provide an
insight into toxicity mechanisms, the molecular details of
these mechanisms remain poorly understood. Nevertheless,
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it is clear that metals and metalloids induce toxicity through
many targets, some of which are metal specific while others
are common for several metals.
Metal responses from a genomic
perspective
Yeast cells respond to metal/metalloid exposure by arresting
cell cycle progression (Pinson et al., 2000; Yen et al., 2005;
Migdal et al., 2008) and by adapting the transcriptome
(Gross et al., 2000; Lyons et al., 2000; Momose & Iwahashi,
2001; Fauchon et al., 2002; Stadler & Schweyen, 2002; Jin
et al., 2003; Haugen et al., 2004; Shakoury-Elizeh et al., 2004;
Thorsen et al., 2007), proteome (Vido et al., 2001; Thorsen
et al., 2007; Pereira et al., 2008) and metabolome (Vido
et al., 2001; Fauchon et al., 2002; Lafaye et al., 2005; Thorsen
et al., 2007). Such responses aim at protecting cells from the
damaging effects of these agents (Fig. 1). In addition, a wide
variety of basal cellular functions contribute to tolerance
acquisition (Haugen et al., 2004; Holland et al., 2007; Dilda
et al., 2008; Jin et al., 2008; Jo et al., 2008; Ruotolo et al.,
2008; Serero et al., 2008; Thorsen et al., 2009). Perhaps the
most comprehensive genome-wide analysis of metal responses to date is the one by Freedman and coworkers; these
authors monitored the transcriptome and ‘deletome’ during
exposure to seven metals including Ag, Cu, Cd, Hg, Zn, Cr
and As (Jin et al., 2008). Most of the other genome-wide
studies mentioned above focused on one/two metals and/or
one particular aspect (transcriptome, proteome, metabolome, deletome) of the metal response. Because in general,
all these studies came to similar conclusions, we will focus
the description below on the findings of Freedman and
colleagues (Jin et al., 2008). A detailed ‘meta-analysis’ of the
transcriptome and deletome during As and Cd exposure was
performed in Thorsen et al. (2009). Transcriptional and
bioinformatics’ analyses pinpointed two groups of genes
responding in a similar way to all metals (Jin et al., 2008).
These genes were termed ‘common metal-responsive’
(CMR) genes; induced CMR genes were enriched in biological processes related to metal ion transport and homeostasis, detoxification of ROS, carbohydrate metabolism,
fatty acid metabolism, polyamine transport and RNA polymerase II transcription, whereas repressed CMR genes were
enriched in biological processes related to polysaccharide
biosynthesis, G-protein signalling, protein targeting and
transport (Jin et al., 2008). In fact, these processes are
similar to those found to be enriched during exposure to
other environmental stresses (Gasch et al., 2000). Non-CMR
genes responded in a less uniform fashion, where some
metals triggered a certain response while others did not. For
example, As and Cu stimulated the expression of genes
involved in energy generation and stress responses; As, Hg,
Cr and Cd triggered the expression of genes in the sulphur
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R. Wysocki & M.J. Tamás
assimilation and GSH biosynthesis pathways; and Cd, Ag,
Hg, Zn and Cr induced genes involved in ribosome biogenesis and tRNA modifications (Jin et al., 2008). The same
authors also analysed the deletome for metal sensitivities
and found that processes related to cell wall integrity, metal
chelation, sequestration of metals in vacuoles and oxidative
stress defence contributed to tolerance acquisition. In contrast to gene expression that involved a common response to
all metals, genes required for tolerance fell into largely
distinct clusters. For instance, mutants sensitive to As were
enriched in processes related to signal transduction, transcriptional regulation, tubulin folding and the secretory
pathway; Cu- and Zn-sensitive genes were enriched in
processes related to vesicle-mediated transport; Cd-sensitive
genes were enriched in processes related to chromatin
modifications, GSH biosynthesis and responses to stress;
Cr-sensitive genes were enriched in processes related to
sulphur amino acid biosynthesis, ubiquitin-dependent protein sorting and trehalose biosynthesis (Jin et al., 2008).
Hence, cells appear to counteract metal toxicity by largely
distinct mechanisms, possibly because the chemical properties of a given metal will influence how it affects cells
(Haugen et al., 2004; Holland et al., 2007; Dilda et al., 2008;
Jin et al., 2008; Jo et al., 2008; Ruotolo et al., 2008; Serero
et al., 2008; Thorsen et al., 2009). This is in analogy to
oxidative stress tolerance, where S. cerevisiae utilizes separate mechanisms for protection against different forms of
ROS (Thorpe et al., 2004). Nevertheless, common metal
responses also exist. The most important example is genes
involved in sulphur assimilation and GSH biosynthesis;
these genes are present in the induced CMR cluster and they
are also required for tolerance to As, Cd, Cr and Cu (Jin
et al., 2008; Thorsen et al., 2009).
Metal uptake pathways and their
regulation
Nonessential and toxic metals and metalloids enter cells on
the basis of molecular mimicry through plasma membrane
permeases and channels evolved for the uptake of essential
metals and other nutrients, such as Fe, Mn, Zn, phosphate,
sulphate and glycerol (Fig. 2). However, all organisms
including yeast have developed mechanisms that can reduce
such influx by downregulating the expression of relevant
transporters at the transcriptional and post-transcriptional
levels and/or by inhibiting their transport activities.
As uptake systems
The arsenate [As(V)] oxyanion is a structural analogue of
inorganic phosphate and is easily taken up through phosphate transporters in most organisms. Phosphate import
into S. cerevisiae is mediated by two high-affinity permeases,
Pho84p and Pho89p, and two low-affinity permeases,
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Yeast metal tolerance
Fig. 2. Transporters mediating the uptake and detoxification of toxic metals in Saccharomyces cerevisiae. See text for an explanation of protein
abbreviations.
Pho87p and Pho90p (Persson et al., 1999; Wykoff & O’Shea,
2001). Deletion of PHO84 and PHO87 confers As(V)
tolerance, suggesting that As(V) enters yeast cells through
these permeases (Bun-ya et al., 1996; Yompakdee et al.,
1996b). Consistently, lack of Pho86p (pho86D), an endoplasmatic reticulum-localized protein involved in trafficking
of Pho84p to the plasma membrane, or Gtr1p (gtr1D), a
cytoplasmic GTPase regulating Pho84p-dependent phosphate transport, also results in increased As(V) tolerance
(Bun-Ya et al., 1992, 1996; Yompakdee et al., 1996a; Lau
et al., 2000). The pho84D mutant also displays enhanced
tolerance to Mn, Co, Zn and Cu, indicating that Pho84p
may mediate the uptake of these metals as well. Moreover,
Pho84p has been shown to play an additional physiological
role as a low-affinity Mn transporter (Jensen et al., 2003)
and has been implicated in Se(IV) tolerance (Pinson et al.,
2004).
In contrast to As(V), the pathway(s) of As(III) entry into
cells remained elusive for a long time. Based on genetic data,
it was proposed that the aquaglyceroporin GlpF mediates
the influx of Sb(III) into Escherichia coli cells (Sanders et al.,
1997). Subsequently, we demonstrated that the aquaglyceroporin Fps1p is the main entrance pathway of As(III) and
Sb(III) into yeast cells (Wysocki et al., 2001). Deletion of
FPS1 results in reduced As(III) uptake and in hypertolerance
to both As(III) and Sb(III). The opposite is observed in cells
harbouring a hyperactive FPS1 allele; these cells display
enhanced As(III) uptake and hypersensitivity to both metalloids (Wysocki et al., 2001). These data clearly establish
Fps1p as the main As(III) and Sb(III) entry pathway. Yeast
cells lacking FPS1 and two additional major metalloid
FEMS Microbiol Rev 34 (2010) 925–951
transporters (acr3Dycf1Dfps1D) has been used to identify
and characterize As(III) and Sb(III) transporting aquaglyceroporins of mammalian and plant origin (Liu et al., 2002;
Bienert et al., 2008; Isayenkov & Maathuis, 2008). Additional studies in bacteria, Leishmania and crop plants (e.g.
rice) have confirmed that aquaglyceroporins are metalloid
entry pathways in probably most organisms (Gourbal et al.,
2004; Meng et al., 2004; Ma et al., 2008). However, it is
important to note that aquaglyceroporins are bidirectional
channels that may also mediate the efflux of metalloids in
certain cases (see later in text). The main form of As(III) in
solution is As(OH)3 (Ramirez-Solis et al., 2004). As(OH)3
structurally resembles Sb(OH)3 and glycerol (Ramirez-Solis
et al., 2004; Porquet & Filella, 2007), the physiological
substrate of E. coli GlpF and S. cerevisiae Fps1p. Hexose
permeases also contribute to As(III) uptake in yeast. It has
been suggested that three As(OH)3 molecules can polymerize to a ring structure similar to hexose sugars that could be
recognized by hexose transporters (Liu et al., 2004).
Fps1p transport activity is regulated, but the mechanisms
are not fully understood. Fps1p has a long cytosolic
N-terminal tail that is important for gating, and it contains
a mitogen-activated protein kinase (MAPK) phosphorylation site (Thr231) (Tamás et al., 1999, 2003; Thorsen et al.,
2006). Deletion of the N-terminal domain or changing
Thr231 into Ala leads to As(III) and Sb(III) sensitivity due
to a high level of unregulated metalloid influx (Wysocki
et al., 2001; Thorsen et al., 2006). We have shown that the
MAPK Hog1p regulates transport through Fps1p; Hog1p
phosphorylates Fps1p on Thr231 and this phosphorylation
reduces Fps1p-mediated transport. Hog1p is activated in
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930
response to As(III) and Sb(III), and cells lacking Hog1p
(hog1D) are very sensitive to both metalloids and display an
increased rate of Fps1p-dependent As(III) uptake (Thorsen
et al., 2006). Two positive regulators of Fps1p activity, the
pleckstrin homology (PH) domain proteins Rgc1p and
Rgc2p (also called Ask10p), were recently identified. Deletion of RGC1 or RGC2 inactivates Fps1p and results in
As(III) tolerance (Beese et al., 2009). However, how these
proteins control Fps1p activity remains unclear. Fps1p is
also regulated at the transcriptional level; addition of As(III)
or Sb(III) to the medium causes a rapid decline in FPS1
mRNA levels (Wysocki et al., 2001). It has been reported
that Fps1p is ubiquitylated and targeted for degradation in a
Hog1p-dependent fashion in acetic acid-exposed cells. Because Fps1p allows the influx of acetic acid, this mechanism
would prevent such influx and toxicity (Mollapour & Piper,
2007). In contrast, Fps1p is not degraded in response to
As(III), suggesting different modes of regulation under
metalloid/acetic acid stress and/or additional functions of
Fps1p during metalloid exposure (Thorsen et al., 2006;
Maciaszczyk-Dziubinska et al., 2010). Interestingly, longterm exposure to As(III) results in the upregulation of FPS1
transcription, whereas the expression of FPS1 from a multicopy plasmid surprisingly increases As(III) and Sb(III)
tolerance (Maciaszczyk-Dziubinska et al., 2010). These data
indicate that Fps1p can mediate both the influx and the
efflux of As(III). In support of this notion, we found that
cells lacking both Fps1p and the As(III) efflux protein Acr3p
were unable to export As(III), while some As(III) export was
evident in the acr3D mutant. Moreover, fps1D cells are As(V)
sensitive even though As(V) is not transported by Fps1p
(Maciaszczyk-Dziubinska et al., 2010). This dual function of
Fps1p in metalloid toxicity (through uptake) and detoxification (through efflux) can be explained by the following
model: As(V) enters cells through phosphate permeases and
is subsequently reduced to As(III). Next, As(III) is actively
extruded as an As(OH)2O anion by Acr3p or it diffuses out
of the cell down the concentration gradient as As(OH)3
through Fps1p. When cells are exposed to As(III), the
activity of Fps1p may be inhibited via its N-terminal domain
in a Hog1p-dependent manner, thereby reducing As(III)
accumulation. However, when the intracellular concentration of As(OH)3 becomes higher than outside the cell, Fps1p
may facilitate As(III) export in concert with Acr3p. Interestingly, to prevent the As(III) exported through Acr3p from
re-entering via Fps1p, cells appear to decrease the extracellular concentration of As(OH)3 by exporting GSH. Extracellular As(III) and GSH form a complex that cannot
enter cells (M. Thorsen, T. Jacobson, R. Vooijs, H. Schat &
M.J. Tamás, unpublished data). Besides Fps1p, aquaglyceroporins from specific bacteria and mammals have also been
implicated in As detoxification (Yang et al., 2005; Carbrey
et al., 2009; McDermott et al., 2010). Moreover, heterolo2010 Federation of European Microbiological Societies
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R. Wysocki & M.J. Tamás
gous expression of several plant aquaporins in yeast resulted
in increased tolerance to As(V), suggesting that these
proteins are bidirectional channels and that they may have
a function in metalloid tolerance (Bienert et al., 2008).
Cd uptake systems
Cd enters cells through proteins involved in the uptake of
essential cations such as Zn (through Zrt1p), Mn (Smf1p,
Smf2p), Fe (Fet4p) and Ca (Mid1p). Zrt1p belongs to the
Zrt- and Irt-like protein (ZIP) family and mediates highaffinity Zn transport (Zhao & Eide, 1996; Eng et al., 1998).
Zrt1p is one of the major pathways through which Cd enters
yeast; cells lacking Zrt1p absorb little Cd from the medium,
while Zn uptake is strongly inhibited by Cd (Gomes et al.,
2002; Gitan et al., 2003). Moreover, Zn-limited cells that
upregulate ZRT1 transcription are more sensitive to Cd.
Consistently, in the presence of high concentrations of Zn
and Cd, Zrt1p is removed from the cell surface to prevent
the uptake of toxic Cd and excess Zn (Gitan et al., 1998;
Gitan et al., 2003). Zrt1p inactivation involves Rsp5pdependent ubiquitylation, followed by endocytosis and
degradation in the vacuole (Gitan & Eide, 2000; Gitan
et al., 2003). Although a putative metal-responsive domain
required for Zn- and Cd-induced Zrt1p ubiquitylation has
been pinpointed, the molecular details of Zrt1p regulation
remain to be established.
Two yeast members of the neutral resistance-associated
macrophage protein (Nramp) family, the Mn transporters
Smf1p and Smf2p, constitute a second pathway of Cd influx.
Smf1p is localized to the plasma membrane and mediates
high-affinity Mn uptake (Supek et al., 1996). The substrates
of Smf1p also include Cd, Co, Cu, Fe and Zn (Supek et al.,
1996; Liu et al., 1997; Chen et al., 1999). Smf2p is also a
major Mn import pathway, although localization of Smf2p
on the cell surface has never been shown (Portnoy et al.,
2000; Luk & Culotta, 2001; Stimpson et al., 2006). Deletion
of SMF1 or SMF2 results in similar levels of Cd tolerance,
while overexpression of these genes leads to Cd sensitivity
(Ruotolo et al., 2008). Hence, Smf1p and Smf2p appear to
contribute equally to Cd accumulation. Smf1p and Smf2p
are ubiquitylated by the ubiquitin ligase Rsp5p that is
targeted to Smf1p and Smf2p by two adaptor proteins:
Bsd2p and Tre1p (Stimpson et al., 2006; Sullivan et al.,
2007). Cells lacking Bsd2p or Tre1p display Cd sensitivity
and elevated Cd uptake due to increased Smf1p and Smf2p
protein levels (Liu et al., 1997; Liu & Culotta, 1999;
Stimpson et al., 2006). In response to Cd, plasma membrane-localized Smf1p is ubiquitylated by Rsp5p and is
rapidly removed from the cell surface by endocytosis (Nikko
et al., 2008). Interestingly, Cd-induced sorting of Smf1p
does not require Bsd2p, but two arrestin-like proteins,
Ecm21p and Crs2p, that recruit Rsp5p to Smf1p. Ecm21p
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Yeast metal tolerance
binds to Smf1p via its constitutively phosphorylated
N-terminal domain, which is not necessary for Bsd2pdependent downregulation (Nikko et al., 2008).
Fet4p is a plasma membrane protein that catalyses lowaffinity Fe import (Dix et al., 1994, 1997), and it can also
mediate the uptake of Cu and Zn (Hassett et al., 2000;
Waters & Eide, 2002). Because Cd and Co inhibit Fet4p
transport activity, it may be involved in the uptake of those
metals as well (Dix et al., 1994; Hassett et al., 2000). The
expression of FET4 is positively regulated at the transcriptional level by the transcription factors Atf1p and Zap1p in
response to low levels of Fe and Zn, respectively (Dix et al.,
1997; Waters & Eide, 2002), and negatively regulated by
Rox1p under aerobic conditions (Jensen & Culotta, 2002;
Waters & Eide, 2002). The lack of Rox1p (rox1D) results in
higher FET4 mRNA levels and Cd sensitivity (Jensen &
Culotta, 2002).
Mid1p, a stretch-activated Ca channel, has been suggested
to be an additional pathway for Cd uptake into yeast. The
lack of Ca uptake in the mid1D mutant is restored by
heterologous expression of the wheat Ca transporter Lct1
and expression of Lct1 sensitizes yeast to Cd due to elevated
Cd accumulation (Clemens et al., 1998). The role of Mid1p
in Cd uptake was recently confirmed by direct transport
assays (A. Gardarin, S. Chedin, J. Aude, E. Godat, P. Catty &
J. Labarre, unpublished data).
Chromate, molybdate and selenate uptake
systems
Based on transport studies in mammalian cells and structural similarities to sulphate, the uptake of Cr(VI), Se(VI) and
molybdate [Mo(VI)] oxyanions via sulphate transporters
has been suggested in yeast (Alexander & Aaseth, 1995;
Tamás et al., 2005). Indeed, deletion of both high-affinity
sulphate permeases, Sul1p and Sul2p, results in enhanced
Cr(VI) and Se(VI) tolerance (Cherest et al., 1997), and
direct transport assays confirmed that Cr(VI) enters yeast
through sulphate permeases (Pereira et al., 2008). A recent
genome-wide screen for Cr-sensitive mutants revealed that
the actin-mediated endocytosis system is involved in Cr
accumulation, and the authors proposed that actin-dependent endocytosis of plasma membrane Cr transporters
might contribute to Cr tolerance. However, such a mechanism does not seem to operate on the level of the Cr(VI)
transporters Sul1p and Sul2p (Holland & Avery, 2009).
Metal detoxification systems and their
regulation
The mechanism that provides the highest level of tolerance
in microorganisms is metal/metalloid removal from the
cytosol through export pathways (Fig. 2). While prokaryotes
possess several export routes, there are only two such
FEMS Microbiol Rev 34 (2010) 925–951
S. cerevisiae pathways characterized in detail: Acr3p (also
called Arr3p) and Pca1p that catalyse the efflux of As(III)
and Cd(II), respectively (Wysocki et al., 1997; Ghosh et al.,
1999; Adle et al., 2007). Because of their importance for
tolerance and high specificity towards a particular metal/
metalloid, Acr3p and Pca1p are tightly regulated by direct
metal-sensing mechanisms (Wysocki et al., 2004; Adle &
Lee, 2008). Transporters located on the vacuolar membrane
also remove metals from the cytosol by catalysing metal
compartmentalization (Fig. 2). In contrast to Acr3p and
Pca1p, these proteins also transport endogenously produced
substrates, their expression is not significantly regulated by
metals and they usually confer a moderate level of tolerance.
The third mode of detoxification involves metal chelation to
specific peptides and proteins, such as GSH, phytochelatins
(PC) and metallothioneins (MT), and the resulting complexes may be recognized as substrates by transporters for
export and/or vacuolar sequestration.
Plasma membrane metal exporters
Acr3p and As tolerance
As(III) export via Acr3p is probably the most important As
detoxification mechanism in S. cerevisiae (Wysocki et al.,
1997; Ghosh et al., 1999). Acr3p is a prototype member of
the arsenical resistance-3 (Acr3) family of transporters,
which belongs to the bile/arsenite/riboflavin transporter
(BART) superfamily (Mansour et al., 2007). Proteins of the
Acr3 family have 10 membrane-spanning helices (Aaltonen
& Silow, 2008; Fu et al., 2009) and are widely distributed in
prokaryotes and fungi, with the exception of fission yeast
Schizosaccharomyces pombe (Wysocki et al., 1997; Maciaszczyk et al., 2004; Mansour et al., 2007). Acr3p homologues
are also present in three lower plant species (Physcomitrella
patens, NCBI accession number XP_001761548; Selaginella
moellendorffii, NCBI accession number ACO57621; and
Pteris vittata, NCBI accession number ACN65413) and in
the animal genome of Danio rerio (NCBI accession number
ACN65413). The ACR3 gene was isolated based on its ability
to confer high-level As tolerance to wild-type S. cerevisiae
when overexpressed (Bobrowicz et al., 1997). Cells lacking
ACR3 are highly As sensitive, hyperaccumulate As and
display impaired As(III) export, while ACR3 overexpression
markedly decreases cytosolic As levels and improves tolerance (Wysocki et al., 1997; Ghosh et al., 1999; Thorsen et al.,
2006). These results establish the role of Acr3p in As(III)
efflux. Thiol chemistry seems to be crucial for transport
through Acr3p; mutagenesis of a conserved cysteine in the
fourth membrane-spanning helix of bacterial and yeast
Acr3p proteins impaired As(III) export and caused metalloid sensitivity (Fu et al., 2009; E. Maciaszczyk-Dziubinska,
D. Wawrzycka, E. Sloma & R. Wysocki, unpublished data).
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All members of the Acr3 family characterized to date confer
tolerance only to As, and not to the related metalloid Sb
(Wysocki et al., 1997; Sato & Kobayashi, 1998; Ghosh et al.,
1999; Fu et al., 2009). In contrast, members of the ArsB
family of transporters, which are ubiquitously present in
bacteria and archaea, but absent in eukaryotes, mediate
export of both metalloids (Rosen, 1999). It has been
proposed that Acr3p may function as a uniporter that
facilitates transport of the As anion As(OH)2O coupled
to the membrane potential (Fu et al., 2009). This model
would explain the specificity of Acr3p towards As(III)
because at a physiologically neutral pH, little Sb(III) anion
would be formed in the cytosol due to the higher pKa value
of Sb(OH)3 compared with that of As(OH)3 (Fu et al.,
2009). Nevertheless, phenotypic analyses of yeast mutants
lacking proteins involved in metalloid uptake and detoxification suggest that Acr3p may transport Sb(III), at
least under specific conditions (Wysocki et al., 2001;
E. Maciaszczyk-Dziubinska, D. Wawrzycka, E. Sloma &
R. Wysocki, unpublished data). The acr3D mutant is highly
sensitive to both As(III) and As(V), but ACR3 overexpression does not lead to high-level As(V) tolerance unless the
ACR2 (also called ARR2) gene is present (Bobrowicz et al.,
1997; Wysocki et al., 1997). Acr2p is an arsenate reductase
that converts As(V) to As(III) (Mukhopadhyay & Rosen,
1998; Mukhopadhyay et al., 2000); hence, As(V) detoxification involves the reduction of As(V) into As(III) for
subsequent export out of cells. The identification of Acr2p
and Acr3p homologues in P. vittata (Chinese brake fern)
indicates that As detoxification involving enzymatic reduction of As(V) to As(III), followed by As(III) export is
conserved from bacteria to plants (Ellis et al., 2006; Salt &
Norton, 2008). Importantly, P. vittata is capable of hyperaccumulating As in the fronds without toxicity and can
therefore potentially be used for phytoremediation of Ascontaminated soils (Ma et al., 2001). Nevertheless, the exact
role of P. vittata Acr2p and Acr3p in this process remains to
be revealed.
Acr3p is regulated at the transcriptional level; the expression of ACR3, and also of ACR2, is tightly controlled by the
transcription factor YAP8 (also called ACR1/ARR1) (Bobrowicz & Ulaszewski, 1998; Wysocki et al., 2004; Ilina et al.,
2008). In fact, ACR2 and ACR3 share a common promoter
and form, together with YAP8, a cluster of As resistance
genes (Bobrowicz et al., 1997). The expression of ACR2 and
ACR3 is very low in the absence of metalloids, but both
genes are simultaneously and robustly induced in a Yap8pdependent manner in the presence of As(III), As(V) or
Sb(III) (Bobrowicz & Ulaszewski, 1998; Bouganim et al.,
2001; Haugen et al., 2004; Menezes et al., 2004; Wysocki
et al., 2004). Yap8p appears to directly sense As(III) by
binding to this metalloid; hence, it couples metalloid sensing
to detoxification by regulating the levels of Acr2p and Acr3p.
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R. Wysocki & M.J. Tamás
Whether Acr2p and Acr3p are also regulated post-translationally is currently not known.
Pca1p and Cd tolerance
Pca1p constitutes the main route of Cd export in S.
cerevisiae and plays a crucial role in Cd tolerance (Adle
et al., 2007). The gene was originally identified in a Cdresistant strain and called CAD2 (Tohoyama et al., 1990),
and it was believed that a gain-of-function mutation within
CAD2/PCA1 (Arg970Gly) caused enhanced Cd efflux and
improved tolerance (Shiraishi et al., 2000). In fact, the PCA1
gene product is not functional in common laboratory
strains because of a Gly970Arg mutation in a conserved
ATP-binding pocket (Adle et al., 2007). Because the CAD2
allele contains Gly at position 970, it actually represents the
wild-type version of Pca1p (Shiraishi et al., 2000). Pca1p
belongs to the ubiquitous P1B-type ATPase super-family
including transporters of a wide range of metals such as Ag,
Cd, Co, Cu, Pb and Zn (Kühlbrandt, 2004). Expression of
wild-type Pca1p confers tolerance by reducing intracellular
Cd levels, establishing this protein as a major detoxification
pathway. Transcription of PCA1 is constitutive, but the
Pca1p protein is not detected under normal conditions due
to rapid turnover. In response to Cd, Pca1p is stabilized and
targeted to the plasma membrane, where it promotes Cd
efflux and tolerance (Adle et al., 2007). The nonfunctional
Pca1p-Gly970Arg is also stabilized in the presence of Cd;
however, it does not reach the plasma membrane, indicating
that the mutation perturbs proper trafficking. Interestingly,
Cu and Ag (but no other metal ions) can also stabilize
Pca1p, and overexpression of either wild-type Pca1p or
Pca1p-Gly970Arg suppresses the Cu toxicity of a Cu-sensitive mutant (ace2D). Pca1p-mediated Cu tolerance involves
an N-terminal cysteine-rich domain, but not its transport
activity. Taken together, Pca1p is a Cd-specific plasma
membrane transporter with the ability to sequester Cu when
overexpressed (Adle et al., 2007).
The mechanism by which Cd regulates Pca1p has been
revealed recently (Adle & Lee, 2008; Adle et al., 2009). The
turnover of many plasma membrane proteins involves
ubiquitin-mediated endocytosis, followed by vacuolar degradation (Leon & Haguenauer-Tsapis, 2009). In contrast,
ubiquitylated Pca1p is targeted for degradation by the
proteasome without first going via the plasma membrane.
Pca1p has a cysteine-rich metal-sensing degradation signal
within its N-terminus that is necessary and sufficient both
for rapid degradation and for Cu- and Cd-induced stabilization of proteins (Adle & Lee, 2008). Metal binding to
cysteines within this domain induces a conformational
change that prevents ubiquitylation and degradation of
Pca1p. Interestingly, Pca1p is targeted for ubiquitylation by
components of the endoplasmic reticulum-associated
FEMS Microbiol Rev 34 (2010) 925–951
933
Yeast metal tolerance
degradation (ERAD) system, like the ubiquitin-conjugating
enzyme Ubc7p and the ubiquitin ligase Doa10p (Adle et al.,
2009). The ERAD pathway serves as a quality control system
involved in the degradation of terminally misfolded secretory proteins (Meusser et al., 2005). Control of Pca1p
turnover by ERAD represents a novel regulatory mechanism
of plasma membrane protein expression and of responses to
metals (Adle et al., 2009).
Other transporters
Some additional plasma membrane transporters have been
implicated in metal detoxification, but little is known about
their regulation or mode of action. Yor1p belongs to the
ATP-binding cassette (ABC) transporter family and is structurally related to the human multidrug resistance-associated
protein MRP1 and yeast Ycf1p (see later in text). The YOR1
gene was identified by its ability to confer oligomycin
resistance when overexpressed and it mediates resistance to
a wide range of chemical compounds including anionic
drugs, fungicides and lipids (Katzmann et al., 1995; Cui
et al., 1996; Decottignies et al., 1998). Deletion of YOR1
results in moderate Cd sensitivity and it probably mediates
Cd efflux in the form of Cd(GS)2 (Cui et al., 1996; Nagy
et al., 2006). Alr1p, which is responsible for Mg uptake into
cells, may also contribute to Cd tolerance. Mutation of the
ALR1 gene results in Cd sensitivity and increased intracellular Cd levels (Kern et al., 2005), but it is not known
whether Alr1p mediates Cd export. Ssu1p is a member of the
major facilitator superfamily and is involved in sulphite
efflux (Park & Bakalinsky, 2000). Overexpression of the
SSU1 gene leads to increased tolerance to Se(IV) (Pinson
et al., 2000), suggesting that Ssu1p mediates Se(IV) export.
However, because deletion of SSU1 causes sensitivity to
sulphite and transcription of SSU1 is induced by sulphite,
but not by Se(IV), it seems that the main function of Ssu1p
is related to sulphite detoxification (Park & Bakalinsky,
2000; Pinson et al., 2000).
Vacuolar sequestration of metals
Transport of metals into vacuoles is a common detoxification mechanism in eukaryotes. In S. cerevisiae, the ABC
transporter Ycf1p represents a major pathway for vacuolar
sequestration of GSH-conjugated metals and xenobiotics, of
endogenously produced toxins such as unconjugated bilirubin and the red pigment formed in adenine biosynthetic
mutants and of free GSH for degradation within the vacuole
(reviewed in Paumi et al., 2009). YCF1 was identified in a
screen for genes conferring increased tolerance to Cd when
overexpressed (Szczypka et al., 1994). Cells lacking YCF1 are
highly sensitive to Cd, Hg, Pb and Sb(III), moderately
sensitive to As(III) (Szczypka et al., 1994; Ghosh et al.,
FEMS Microbiol Rev 34 (2010) 925–951
1999; Wysocki et al., 2001; Gueldry et al., 2003; Song et al.,
2003; Preveral et al., 2006), but grow normally in the
presence of other metals/metalloids (Preveral et al., 2006).
Ycf1p catalyses active transport of As(GS)3, Cd(GS)2 and
Hg(GS)2 into vacuoles in vitro, while the presence of Sb(III)
inhibits Ycf1p-mediated transport of GSH conjugates (Li
et al., 1997; Ghosh et al., 1999; Gueldry et al., 2003).
Together, these data establish the role of Ycf1p-mediated
transport of metal–GSH conjugates for metal tolerance.
How Ycf1p-mediated detoxification is regulated is not
well understood. YCF1 is transcribed in the absence of
metals and its expression is only moderately increased by
Cd, Hg, As(III), Sb(III) and Se(IV) (Li et al., 1997; Pinson
et al., 2000; Sharma et al., 2002; Gueldry et al., 2003;
Wysocki et al., 2004). In fact, strong induction of YCF1
expression is only observed when the transcriptional regulator Yap1p is overexpressed (Wemmie et al., 1994; Sharma
et al., 2002) or in mutants that hyperaccumulate As in the
cytosol (Wysocki et al., 2004). Ycf1p is subjected to posttranslational control at the level of proteolytic processing,
intracellular trafficking and phosphorylation (Mason &
Michaelis, 2002; Mason et al., 2003; Eraso et al., 2004; Paumi
et al., 2008). Ycf1p is positively modulated by phosphorylation and by the guanine exchange factor Tus1p. However,
mutating the phosphorylated residues or deleting TUS1
causes only moderate Cd sensitivity (Eraso et al., 2004;
Paumi et al., 2007). Ycf1p is also negatively regulated by
phosphorylation; mutation of Ser251 increases both its
transport capacity in vitro and Cd tolerance in vivo. An
integrated membrane yeast two-hybrid screen revealed two
candidate kinases, Cka1p and Hal5p, which may act as the
negative regulators of Ycf1p (Paumi et al., 2008). Nevertheless, further studies are required to establish whether and
how Ycf1p activity is modulated during metal exposure.
Genetic and biochemical studies implicated additional
vacuolar transporters in metal tolerance. Two Ycf1p paralogues, Bpt1p and Vmr1p, play a minor role in Cd
detoxification (Sharma et al., 2002; D. Wawrzycka & A.
Goffeau, unpublished data). Deletion of BPT1 sensitizes
cells to Cd only in the absence of YCF1, and the expression
of BPT1 is not induced by Cd (Sharma et al., 2002).
Similarly, the lack of Vmr1p causes slight sensitivity to Cd
(D. Wawrzycka & A. Goffeau, unpublished data). Sequestration of several divalent metal cations, including Cd and
Co, into vacuoles requires the presence of Zrc1p and Cot1p,
which play important roles in Zn homeostasis (MacDiarmid et al., 2000; 2002). Both transporters belong to the
cation diffusion facilitator (CDF) family (Paulsen & Saier,
1997) and share the ability to transport Zn and Co (Conklin
et al., 1992, 1994). Zrc1p is also likely to transport Cd and
Ni as these metals inhibit Zrc1p-dependent Zn uptake into
vacuoles (MacDiarmid et al., 2002). In addition, overexpression of COT1 and ZRC1 confers resistance to
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934
rhodium (Rh) and Cd, respectively (Kamizono et al., 1989;
Conklin et al., 1992). Hence, these transporters may contribute to Cd and Co tolerance. Finally, cells lacking the
vacuolar P5-type ATPase Ypk9p are slightly sensitive to Cd,
Mn, Ni and Se(II) (Gitler et al., 2009; Schmidt et al., 2009).
However, how this protein contributes to metal tolerance
remains to be established.
Cd tolerance in S. pombe involves the ABC transporter
Hmt1, which is localized to the vacuolar membrane (Ortiz
et al., 1992). Because fission yeast, like plants, uses PC to
chelate metals (see later in text), the major substrate for
Hmt1 seemed to be Cd–PC complexes (Ortiz et al., 1995).
However, this two-step model involving the formation of
metal–PC complexes, followed by vacuolar accumulation
via Hmt1-like transporters thought to operate in fission
yeast and plants, has been questioned recently (Preveral
et al., 2009; Sooksa-Nguan et al., 2009). Firstly, overexpression of S. pombe Hmt1 confers high-level Cd tolerance to S.
pombe cells devoid of the enzyme catalysing PC biosynthesis,
and also in budding yeast and bacteria that naturally lack PC
(Preveral et al., 2009). Secondly, PC-deficient fission yeast
display increased sensitivity to As, Cd and Hg, while cells
lacking Hmt1 are sensitized only to Cd. Thirdly, Hmt1
proteins appear to transport mainly GSH-conjugated Cd,
but little or no metal–PC complexes (Preveral et al., 2009;
Sooksa-Nguan et al., 2009). Thus, vacuolar sequestration of
metal–GSH conjugates mediated by ABC transporters appears to be conserved from bacteria to humans. In addition,
chelation of metals by PC in the cytosol represents a distinct
mode of detoxification.
Metal chelation by MT and PC
Low-molecular-weight cysteine-rich proteins and peptides
such as GSH, MT and PC are important contributors to
metal detoxification. In S. cerevisiae, there are two types of
MT, Cup1p and Crs5p, which differ structurally and functionally. Cup1p is a 53-amino-acid-long polypeptide, it has
limited homology to mammalian MT and it binds Cu, Cd
and Zn (Winge et al., 1985). In the absence of metals, CUP1
expression is negligible, but is strongly induced by high Cu
levels (Thiele & Hamer, 1986). Cu tolerance can be achieved
by genomic CUP1 gene amplification (Fogel & Welch, 1982;
Liti et al., 2009) and cup1D cells are sensitive to Cu, but not
to other metals/metalloids (Ecker et al., 1986; R. Wysocki &
M.J. Tamás, unpublished data). Hence, the main physiological role of Cup1p appears to be Cu detoxification. Because
high expression of CUP1 leads to enhanced Cd tolerance, it
is possible that Cup1p also protects cells from Cd toxicity
(Ecker et al., 1986). Crs5p is a 69-residue protein, is more
cysteine-rich than Cup1p and shows high similarity to MT
from other organisms (Culotta et al., 1994). Crs5p is
constitutively expressed, but in the presence of high Cu
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R. Wysocki & M.J. Tamás
levels, its expression is elevated, albeit to a lesser extent than
CUP1. The crs5D mutant is slightly Cu sensitive (Jensen
et al., 1996). Recently, Crs5p was shown to bind Cu, Cd and
especially well to Zn. Moreover, the protein seems to be
essential for yeast viability during mixed Cu/Zn overload.
Hence, Crs5p might be a Zn-thionein with a physiological
function in Zn homeostasis (Pagani et al., 2007). Likewise,
the S. pombe metallothionein Zym1 sequesters Zn, while
cells lacking Zym1 are sensitive to Zn and Cd (Borrelly et al.,
2002). In sum, yeast MT are mainly involved in Cu and Zn
homeostasis, with a limited role in Cd tolerance.
PC are peptides found in all eukaryotic kingdoms and in
some prokaryotes, and they are composed of (g-Glu-Cys)nGly (PCn; n = 2–11) repeats. These peptides are synthesized
from GSH by the enzyme PC synthase (PCS). PC constitute
a major pathway of metal detoxification in fission yeast and
plants, especially for Cd and As detoxification (Clemens,
2006). PC biosynthesis is stimulated by a wide range of
metals and metalloids, including Cd, Cu, Ag, Hg, Zn and As,
while deletion of the S. pombe PCS gene results in As(III),
Cd and Cu sensitivity (Clemens et al., 1999, 2001; Ha et al.,
1999; Wysocki et al., 2003). The formation of Ag–, Cd–, Cu–
and As–PC complexes has been demonstrated in vitro
(Maitani et al., 1996; Schmoger et al., 2000). Hence, PC
appears to protect cells by chelating metals, thereby reducing
their reactivity and toxicity. Importantly, heterologous expression of PCS in S. cerevisiae, or in mutants lacking
functional vacuoles, can confer Cd, As(III) and Sb(III)
tolerance (Clemens et al., 1999; Wysocki et al., 2003). Thus,
chelation of metals by PC in the cytosol is sufficient for
improving metal tolerance. As mentioned above, the
S. cerevisiae genome does not encode a PCS homologue.
Nevertheless, the formation of PC2 has been detected in this
yeast in response to Cd, Cu and Zn (Kneer et al., 1992). PC2
formation involves two vacuolar carboxypeptidases: CPY
and CPC (Wünschmann et al., 2007). However, cells lacking
these enzymes are not Cd sensitive despite an inability to
produce PC2. Thus, naturally produced PC in S. cerevisiae
does not appear to contribute to metal tolerance to any large
extent.
Other detoxification systems
As(V) tolerance involves the arsenate reductase Acr2p;
ACR2 deletion sensitize cells only to As(V) and purified
Acr2p catalyses the reduction of As(V) into As(III) using
GSH and glutaredoxin as electron donors (Mukhopadhyay
& Rosen, 1998; Mukhopadhyay et al., 2000). Acr2p shows no
similarity to bacterial ArsC arsenate reductases; instead,
Acr2p contains a conserved protein tyrosine phosphatase
signature HisCys(X)5Arg motif (Fauman et al., 1998). Acr2p
has no phosphatase activity, but uses the HisCys(X)5Arg
motif as the catalytic centre for As(V) reduction
FEMS Microbiol Rev 34 (2010) 925–951
935
Yeast metal tolerance
(Mukhopadhyay & Rosen, 2001). Interestingly, Acr2p can
easily be converted into a phosphatase merely by substituting three amino acids (Mukhopadhyay et al., 2003), and
LmACR2 from Leishmania major exhibits both arsenate
reductase and protein tyrosine phosphatase activities (Zhou
et al., 2006). Hence, Acr2p arsenate reductases probably
evolved from protein tyrosine phosphatases as a distinct
protein family (Mukhopadhyay et al., 2003).
Sulphur and GSH metabolism
Sulphur assimilation and GSH biosynthesis are essential for
all organisms. In S. cerevisiae, extracellular sulphate is taken
up by sulphate transporters and reduced through the
assimilation pathway to yield sulphide. Sulphide can then
either go through the methyl cycle or into the cysteine/GSH
biosynthesis pathway (Fig. 3). Hence, assimilated sulphur
will either be incorporated into the sulphur-containing
amino acids methionine and cysteine or into the lowmolecular-weight thiol molecules S-adenosylmethionine
and GSH. Transcription of the genes encoding the above
activities is regulated by the transcriptional activator Met4p,
but in response to metals, Met4p acts in concert with Yap1p
to regulate GSH synthesis (see later in text). An essential
function of the sulphur pathway is its involvement, through
S-adenosylmethionine, in the biosynthesis of polyamines
and biotin and most transmethylation reactions in the cell
(Thomas & Surdin-Kerjan, 1997). Another essential function of this pathway is GSH biosynthesis. GSH is the main
redox buffer of the cell and it serves as an electron donor for
many enzymes. GSH is also a key factor in the defence
against oxidative stress and metal toxicity (Thomas &
Surdin-Kerjan, 1997; Grant, 2001), and it contributes to
metal detoxification in several ways. Firstly, GSH can bind to
metals and the resulting complex is a substrate for proteins
that mediate vacuolar sequestration. Secondly, GSH protects
cells against metal-induced oxidation. Thirdly, GSH may
bind to reactive sulphydryl groups on proteins (protein
glutathionylation), thereby shielding them from irreversible
metal binding and/or oxidative damage (Grant, 2001; Pompella et al., 2003). While the first two mechanisms are well
characterized, less is known about the role of protein
glutathionylation in metal tolerance. Recently, we discovered a fourth detoxification function for GSH involving
extracellular metal chelation. As(III)-exposed yeast cells
export significant amounts of GSH and extracellular GSH
forms a complex with As(III) outside cells. This complex
does not readily enter cells; consequently, cells that produce
and export GSH accumulate less As in the cytosol and grow
better during As(III) exposure (M. Thorsen, T. Jacobson,
R. Vooijs, H. Schat & M.J. Tamás, unpublished data).
The expression of genes in the sulphate assimilation and
GSH biosynthesis pathways is stimulated by As(III), Cd, Hg
and Cr(VI) (Momose & Iwahashi, 2001; Vido et al., 2001;
Fauchon et al., 2002; Haugen et al., 2004; Thorsen et al.,
2007; Jin et al., 2008). The levels of the corresponding
enzymes also increase in As(III)-, Cd- and Cr(VI)-exposed
cells (Vido et al., 2001; Thorsen et al., 2007; Pereira et al.,
2008). In response to As(III) and Cd, the cells also boost
sulphur metabolite pools, the GSH synthesis rate, as well as
Fig. 3. Sulphur and GSH metabolism in Saccharomyces cerevisiae. The sulphur pathway can be divided into three parts: the sulphate assimilation
pathway, the methyl cycle and the branch leading to cysteine and GSH synthesis. See text for details.
FEMS Microbiol Rev 34 (2010) 925–951
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936
the flux in the GSH pathway (Vido et al., 2001; Fauchon
et al., 2002; Lafaye et al., 2005; Thorsen et al., 2007). In the
case of Cd and As exposure, cells appear to redirect sulphur
metabolism by routing a large part of the assimilated
sulphur into GSH biosynthesis at the expense of sulphur
incorporation into proteins (Fauchon et al., 2002; Thorsen
et al., 2007). Although Cr(VI) also triggers enhanced levels
of sulphur pathway enzymes, it strongly reduces sulphate
assimilation and sulphur metabolite pools (Pereira et al.,
2008). This is in clear contrast to the response to As and Cd;
these metals elicit increased metabolite pools and pathway
flux. Cr(VI) competitively inhibits sulphate uptake and it
may also affect a step in the sulphur assimilation pathway,
possibly at the level of the ATP-sulphurylase Met3p (Pereira
et al., 2008). Hence, whereas As(III)- and Cd-exposed cells
activate the sulphur/GSH pathway despite increased levels of
metabolite pools, Cr(VI)-triggered expression of these genes
is probably caused by sulphur starvation (Pereira et al.,
2008). Hence, proteome and metabolome data can display a
positive (Vido et al., 2001; Fauchon et al., 2002; Lafaye et al.,
2005; Thorsen et al., 2007) or a negative (Lafaye et al., 2005;
Pereira et al., 2008) correlation within the same pathway
depending on the condition that the cell encounters. The
above examples underscore that careful analysis of the
transcriptome, proteome and metabolome is necessary to
make firm conclusions about how metals impact metabolism and/or cause toxicity.
Other metals also interfere with the sulphate assimilation
pathway. Selenate [Se(VI)], like chromate, probably enter
cells through sulphate transporters, and Se(VI)-resistant
mutants encode components of the sulphate assimilation
pathway (Breton & Surdin-Kerjan, 1977; Cherest et al.,
1997). These data suggest that the conversion of Se(VI) into
Se(III) may cause toxicity. Alternatively, a sulphur metabolite might accumulate in the mutants as a consequence of the
lacking enzyme activity, and cause repression/inhibition of
sulphate transporters and hence resistance to the toxic
sulphate analogue Se(VI). Similarly, the metalloid tellurite
[Te(III)] is reduced to elemental tellurium [Te(0)] via the
sulphate assimilation pathway. Deletion of the sulphite
reductases MET10 or MET5 results in a lack of Te(III)
reduction and in enhanced Te(III) tolerance (L. Ottosson,
K. Logg, L. Tong, P. Sunnerhagen, M. Käll, A. Blomberg &
J. Warringer, unpublished data). Hence, in these cases, the
sulphur assimilation pathway is mediating metal toxicity.
Yeast cells exposed to Cd or Cr(VI), or undergoing
sulphur starvation, trigger a so-called sulphur-sparing response (Fauchon et al., 2002; Pereira et al., 2008). This
response was first described for Cd-treated cells and involves
induction of several isoenzymes with functions in carbohydrate metabolism, for example pyruvate decarboxylase,
aldehyde dehydrogenase and enolase. The induced isoenzymes have a lower content of sulphur-containing amino
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R. Wysocki & M.J. Tamás
acids compared with those expressed under nonstress conditions, while expression of the latter (sulphur-rich) enzymes is repressed. The isoenzyme switch was linked to the
necessity to spare sulphur to allow cells to strongly enhance
GSH levels during Cd exposure (Fauchon et al., 2002).
Optimal sulphur sparing also involves a decrease in the
global protein synthesis rates coupled to the reduced sulphur amino acid composition of the newly synthesized
proteome. These responses might allow an overall sulphur
amino acid saving of up to 30% (Fauchon et al., 2002; Lafaye
et al., 2005). The transcription factor Met4p regulates the
isoenzyme switch and plays a major role in the sulphursparing response, indicating that the same regulator controls
GSH synthesis and the mechanisms that save sulphur for
enhanced GSH production (Fauchon et al., 2002). This
regulation emphasizes the importance of sulphur sources in
detoxification and indicates that a selective pressure may act
on the atomic composition of proteins (Baudouin-Cornu
et al., 2001). As mentioned above, Cr(VI) and sulphur
starvation also trigger the sulphur-sparing response. However, in these cases, the cellular pools of sulphur metabolites
and GSH are significantly reduced, and the sulphur-sparing
response might be a consequence of sulphur deprivation
(Lafaye et al., 2005; Pereira et al., 2008) rather than a need to
spare sulphur for incorporation into GSH.
Cd detoxification in S. pombe involves GSH and PC;
however, the levels of the enzymes of the sulphate assimilation, GSH and PC biosynthesis pathways do not increase in
the presence of Cd (Chen et al., 2003; Bae & Chen, 2004).
Despite this, PC synthesis is elevated because binding of Cd
to PCS strongly activates the enzyme (Grill et al., 1991;
Vatamaniuk et al., 2000; Maier et al., 2003). Sulphide
synthesis is also increased in S. pombe (Chen et al., 2003;
Bae & Chen, 2004) and sulphide participates in the production of the high-molecular-weight PC–Cd–S complex that
has a high Cd-binding capacity (Ow et al., 1994). A similar
mechanism has been described in Candida glabrata (Dameron et al., 1989). Interestingly, PC-based detoxification of Cd,
As(III) and Sb(III) seems to be more efficient than the GSHbased detoxification mechanism operating in S. cerevisiae
because the expression of S. pombe or Arabidopsis thaliana
PCS in S. cerevisiae improved its tolerance to these metals
(Clemens et al., 1999; Wysocki et al., 2003).
Signalling and transcriptional regulation
Signalling proteins and transcriptional regulators are key
mediators of the cell’s response to metals (Fig. 4). These
proteins sense the presence of metals, and regulate various
tolerance and detoxification systems. In recent years, several
such regulators have been identified, and the molecular
mechanism by which they are activated and contribute to
tolerance is starting to emerge.
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Yeast metal tolerance
Fig. 4. Signalling proteins and transcriptional regulators involved in metal tolerance. See text for details.
Yap1p: protection against oxidants and metals
Yap1p (yeast AP-1) is one of eight AP-1-like transcription
factors (Yap1p–Yap8p) in S. cerevisiae. These proteins
contain a basic leucine zipper (bZIP) DNA-binding
domain and a number of conserved cysteine residues
(Moye-Rowley et al., 1989; Fernandes et al., 1997; Toone
et al., 2001). In Yap1p, these cysteines are clustered into two
domains – a C-terminal cysteine-rich domain (cCRD)
composed of residues Cys598, Cys620 and Cys629 and an
N-terminal cysteine-rich domain (nCRD) composed of
residues Cys303, Cys310 and Cys315 – and they are
important for Yap1p regulation (Toone et al., 2001; D’Autreaux & Toledano, 2007). Yap1p mediates the cell’s
response to peroxides and other oxidants, chemicals with
electrophilic properties, and metals by controlling expression of about 70 genes. The majority of those genes encode
proteins that maintain a favourable redox balance in the
cell, enzymes involved in detoxification of ROS and proteins conferring metal and drug resistance (Lee et al., 1999;
Gasch et al., 2000; Haugen et al., 2004; Thorsen et al.,
2007). Cells lacking YAP1 fail to properly induce the
expression of those genes and are sensitive to a broad range
of oxidants, chemical agents and metals. Yap1p regulates
target-gene expression principally by binding to the socalled Yap1p recognition element (YRE) with the consensus
sequence TT/GAC/GTAA (Kuge & Jones, 1994; Wu &
Moye-Rowley, 1994; Fernandes et al., 1997; Harbison
et al., 2004; Tan et al., 2008), but it may also act through
alternative YRE sequences (He & Fassler, 2005).
FEMS Microbiol Rev 34 (2010) 925–951
Yap1p can be activated by a multitude of stress signals
including peroxides, diamide (Kuge & Jones, 1994), menadione (Stephen et al., 1995; Stephen & Jamieson, 1997), the
electrophiles diethylmaleate (Kuge & Jones, 1994), benomyl
and MMS (Nguyen et al., 2001), and the metals Cd (Hirata
et al., 1994; Stephen & Jamieson, 1997), As(III) (Menezes
et al., 2004; Wysocki et al., 2004), Sb(III) (Wysocki et al.,
2004), Se(III) (Azevedo et al., 2003) and Hg (Westwater
et al., 2002). Moreover, the yap1D mutant displays sensitivity to these agents. Importantly, all these signals control
Yap1p through regulated nuclear export by stress-induced
post-translational modifications. In the absence of stress,
Yap1p is mainly localized to the cytosol due to rapid nuclear
export by the nuclear export receptor Crm1p. Crm1p
interacts with a nuclear export signal (NES) embedded
within the Yap1p cCRD (Kuge et al., 1997, 1998; Yan et al.,
1998). Upon exposure to stress signals, the Yap1p–Crm1p
interaction is lost due to redox- or chemical-dependent
modifications of Yap1p cysteines, resulting in the nuclear
accumulation of Yap1p (Kuge et al., 1998; Yan et al., 1998).
H2O2-induced Yap1p activation involves the formation of
an intramolecular disulphide bond between the cCRD
Cys598 and the nCRD Cys303 (Delaunay et al., 2000), and
possibly also between Cys310 (nCRD) and Cys629 (cCRD)
(Wood et al., 2003). Disulphide linkage between these
N- and C-terminal cysteines triggers a conformational
change that masks the Yap1p NES and results in the nuclear
accumulation of the protein. Reduction of these disulphide
bonds causes structural modifications that lead to NES
exposure and redistribution of Yap1p to the cytosol (Wood
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et al., 2004). Yap1p activation by H2O2 involves Gpx3p (also
called Orp1p), which acts as the actual H2O2 sensor
(Delaunay et al., 2002; Paulsen & Carroll, 2009), and a
second protein Ybp1p (Veal et al., 2003; Gulshan et al.,
2004), whose function is not understood. The Gpx3p–
Yap1p H2O2 sensor operates as a cysteine-redox relay, where
Gpx3p–Cys36 senses the H2O2 signal and oxidizes to a
Cys–SOH. Oxidized Gpx3p transduces this signal to Yap1p
by engaging the latter into a Cys36–Cys598 intermolecular
disulphide, which is then converted to the intramolecular
Cys303–Cys598 disulphide of active Yap1p (Delaunay et al.,
2002; Paulsen & Carroll, 2009). Yap1p oxidation is
rapid and the formation of interdomain disulphides results
in an active form of Yap1p that is more resistant to
reduction/deactivation by thioredoxin (Izawa et al., 1999;
Delaunay et al., 2000; Carmel-Harel et al., 2001; Okazaki
et al., 2007).
Although metals and chemicals also activate Yap1p by
regulating its nuclear export, these compounds are sensed
through a distinct mechanism that does not involve interCRD disulphide bonds and bypass the requirement of
Gpx3p and Ybp1p (Kuge et al., 1997; Delaunay et al., 2000;
Azevedo et al., 2003; Veal et al., 2003; Gulshan et al., 2004).
The electrophile N-ethylmaleimide activates Yap1p by the
covalent modification of cCRD Cys598, Cys620 and Cys629
(Azevedo et al., 2003) while Yap1p activation by diamide
involves the formation of intra-CRD disulphide bonds
between either one of Cys598, Cys620 and Cys629 (Kuge
et al., 2001). Cd- and Se(III)-triggered activation of Yap1p
proceeds through the cCRD (Azevedo et al., 2003), whereas
As(III)-triggered activation may be more complex. In the
latter case, Yap1p mutants with modified cCRD cysteines or
nCRD cysteines together with modified Cys620 displayed
perturbed nuclear retention and reduced ability to confer
As(III) tolerance (Wysocki et al., 2004). The exact details by
which metals interact with or modify Yap1p, for example by
direct binding or through oxidative modifications, and
whether those modifications disrupt the Yap1p–Crm1p
interaction, remain to be revealed.
Interestingly, Yap1p appears to act together with other
transcription factors to orchestrate responses to various
stress conditions. Yap1p controls the oxidative stress response in cooperation with Skn7p (Lee et al., 1999); it
regulates GSH biosynthesis together with Met4p (Wheeler
et al., 2003; Thorsen et al., 2007); it acts together with Pdr1p
and Pdr3p to regulate multidrug resistance pathways
(Wendler et al., 1997); and it is involved in regulating the
expression of RPN4 (Owsianik et al., 2002; Haugen et al.,
2004), a transcription factor controlling proteasomal gene
expression. Together, these transcription factors form an
interconnected transcriptional network that mediates the
cellular response to the toxic and damaging effects of
environmental stress agents.
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R. Wysocki & M.J. Tamás
Many different fungi possess functional homologues of
Yap1p, for example S. pombe Pap1 (Toone et al., 2001) and
Candida albicans Cap1 (Alarco & Raymond, 1999). Similar
to Yap1p, S. pombe Pap1 is activated by H2O2-induced
disulphide bond formation involving a peroxiredoxin,
Tpx1, that transfers the redox signal to and hence activates
Pap1 (Vivancos et al., 2004, 2005). Moreover, Pap1 is
required for Cd and As(III) tolerance because deletion of
the pap1 gene results in sensitivity to these metals (Rodriguez-Gabriel & Russell, 2005; Kennedy et al., 2008). The
mechanisms by which metals activate Pap1 are unknown.
Yap8p: an arsenite-sensing transcription factor
Yap8p (also called Acr1p/Arr1p) is another metal-responsive
AP-1-like transcription factor. In contrast to Yap1p, which
protects cells in response to many stress conditions, Yap8p is
exclusively required for As tolerance. Yap8p is necessary for
As(III)-induced expression of ACR2 and ACR3, which
appear to be its only gene targets (Wysocki et al., 2004; Ilina
et al., 2008). The shared ACR2-ACR3 promoter contains an
extended pseudopalindromic YRE (TGATTAATAATCA) sequence that is recognized by Yap8p, but not by Yap1p. We
have shown that this sequence is essential for Yap8p binding
and for As(III)-induced ACR2 and ACR3 expression (Wysocki et al., 2004; Ilina et al., 2008). Yap8p is not regulated at
the level of localization; instead, it resides in the nucleus
bound to the ACR2-ACR3 promoter both in untreated and
in As(III)-exposed cells (Wysocki et al., 2004; Di & Tamás,
2007). Yap8p is regulated by the ubiquitin–proteasome
pathway; Yap8p is present in low levels in untreated cells
due to degradation, whereas Yap8p is stabilized in As(III)exposed cells and stimulates enhanced transcription of its
target genes (Di & Tamás, 2007). Yap8p degradation involves
the ubiquitin-conjugating enzyme Ubc4p while the ubiquitin ligase that acts on Yap8p remains to be identified. How
As(III) activates Yap8p is not fully understood. Proper
Yap8p function requires cysteine residues that are conserved
in Yap1p and other fungal AP-1 proteins; the mutation of
Yap8p Cys132, Cys137 or Cys274 affects Yap8p stabilization,
disrupts As(III)-triggered ACR3 expression and results in As
sensitivity (Menezes et al., 2004; Wysocki et al., 2004; Di &
Tamás, 2007). Based on these data, we proposed that Yap8p
might be activated by direct binding to As(III) (Wysocki
et al., 2004; Di & Tamás, 2007). Indeed, analysis with X-ray
spectroscopy revealed that As(III) binds to purified Yap8p
with high affinity in an S3 site, pointing to the involvement
of three cysteine residues. A Yap8p mutant lacking Cys132
and Cys274 shows dramatically reduced As(III) binding
ability, indicating that these are two of the three cysteines
comprising the three-coordinate binding site (J. Yang & B.P.
Rosen, unpublished data). Hence, Yap8p can be viewed as a
sensor protein that efficiently couples As(III) sensing to
FEMS Microbiol Rev 34 (2010) 925–951
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Yeast metal tolerance
detoxification. Nevertheless, the mechanism by which
As(III) binding enables Yap8p to trigger ACR2-ACR3 expression remains to be unveiled.
Yap8p homologues are only present in the genomes of three
closely related fungal species: Saccharomyces paradoxus, Saccharomyces kudriavzevii and Kluyveromyces lactis (Ilina et al.,
2008). Kluyveromyces lactis possesses a functional Yap8p orthologue; this protein can fully complement the As(III) sensitivity
of S. cerevisiae yap8D, and the K. lactis yap8D mutant is As
sensitive. Interestingly, K. lactis Yap8p may play an additional
role in mediating tolerance to Cd and peroxides (J. Veide-Vilg,
E. Maciaszczyk-Dziubinska, E. Sloma, R. Wysocki & M.J.
Tamás, unpublished data). Whether this additional function
of K. lactis Yap8p is due to a different sensing mechanism and/
or DNA-binding capacity compared with that of S. cerevisiae
Yap8p is currently under investigation.
Met4p: a regulator of sulphur and GSH
metabolism
The bZIP protein Met4p is the principal transcriptional
activator of the sulphur assimilation and GSH biosynthesis
pathways (Fig. 3). Met4p controls the expression of its gene
targets in response to variations in the intracellular pool of
an organic sulphur compound, possibly cysteine (Thomas &
Surdin-Kerjan, 1997; Hansen & Johannsen, 2000; Menant
et al., 2006), in response to metals such as As(III), Cd and
Cr(VI), and it also controls the aforementioned sulphursparing programme (Fauchon et al., 2002; Thorsen et al.,
2007; Pereira et al., 2008). Met4p is unable to bind to DNA
directly; instead, it is recruited to target promoters by the
DNA-binding proteins Met31p, Met32p and Cbf1p. Another cofactor, Met28p, stabilizes the DNA-bound Met4pcontaining complexes (Thomas & Surdin-Kerjan, 1997).
The Met4p–Cbf1p–Met28p complex is targeted to the
regulatory sequence CACGTGA by the centromere-binding
factor Cbf1p, while the Met4p–Met28p–Met31p–Met32p
complex is targeted to the DNA sequence AAACTGTGGC
by the redundant Met31p–Met32p proteins (Blaiseau et al.,
1997; Kuras et al., 1997; Thomas & Surdin-Kerjan, 1997;
Blaiseau & Thomas, 1998). The core Met4p regulon consists
of about 45 genes; the expression of the core genes requires
Met31p or Met32p, whereas the loss of Cbf1p affects only a
subset of gene targets (Lee et al., 2010).
Met4p regulation involves Met30p, the F-subunit of the
SCFMet30 ubiquitin ligase complex that targets Met4p for
ubiquitylation and inactivation. Interestingly, ubiquitylated
Met4p can encounter different fates depending on the
growth condition; it can be targeted by the 26S proteasome
for degradation or it can be maintained in its ubiquitylated
form without degradation (Kaiser et al., 2000; Rouillon
et al., 2000; Kuras et al., 2002; Chandrasekaran et al., 2006;
Flick et al., 2006). When methionine is added back to
FEMS Microbiol Rev 34 (2010) 925–951
sulphur-starved cells grown in minimal medium, SCFMet30
targets Met4p for poly-ubiquitylation and degradation
(Rouillon et al., 2000; Kuras et al., 2002). In rich medium
where sulphur compounds are abundant, Met4p is oligoubiquitylated (one to four ubiquitin moieties), but not
degraded. It has been shown that a ubiquitin-binding motif
within Met4p protects the protein from degradation and
that oligo-ubiquitylated Met4p fails to form functional
transcriptional complexes with its auxiliary factors (Kaiser
et al., 2000; Kuras et al., 2002; Chandrasekaran et al., 2006;
Flick et al., 2006). In response to Cd, both mechanisms of
Met4p inactivation described above are lost, resulting in the
rapid induction of Met4p-dependent genes. Cd inhibits
SCFMet30 activity by triggering the dissociation of Met30p
from the ubiquitin ligase complex. This is followed by a
deubiquitylation step that removes ubiquitin from Met4p,
thereby fully restoring Met4p activity (Barbey et al., 2005;
Yen et al., 2005). However, neither the mechanism by which
Cd triggers dissociation of Met30p from the ubiquitin ligase
complex nor the identity of the deubiquitylating enzyme
that acts on Met4p is known. Similar effects on Met4p
ubiquitylation have been observed with As(III) (Yen et al.,
2005), which is consistent with the importance of Met4p for
As(III)-induced expression of sulphur/GSH genes (Thorsen
et al., 2007). However, this mode of Met4p regulation does
not seem to apply in response to Co, Ni and Pb (Yen et al.,
2005). Interestingly, fully activated Met4p triggers a cell
cycle arrest (Patton et al., 2000; Su et al., 2005). The
observation that Cd- and As(III)-treated cells arrest cell
cycle progression (Yen et al., 2005; Migdal et al., 2008)
suggests a possible role for Met30p–Met4p in this control.
Schizosaccharomyces pombe induces the expression of
genes encoding transporters of sulphur compounds in
response to Cd (Chen et al., 2003) via the bZIP transcription
factor Zip1 (Harrison et al., 2005). Zip1 is regulated by the
SCFpof1 ubiquitin ligase by a mechanism similar to how
SCFMet30 regulates Met4p in S. cerevisiae involving Cdmediated Zip1 stabilization and induced expression of Zip1
gene targets (Harrison et al., 2005). Remarkably, this mechanism of regulation seems to be conserved in mammals;
the SCF ubiquitin ligase complex Keap1/Cul3/Rbx1 controls
degradation of the bZIP transcription factor Nrf2. This
factor is stabilized by Cd and oxidative stress (Stewart et al.,
2003; Kobayashi et al., 2004), and Nrf2 also controls GSH
biosynthesis (Chan & Kwong, 2000; Sun et al., 2005),
indicating an evolutionary conservation of this feature
among eukaryotes.
Rpn4p: regulating proteasomal degradation of
damaged proteins
Many metals may interfere with enzyme/protein activity, and
the ubiquitin–proteasome pathway provides a mechanism
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940
to remove damaged or nonfunctional proteins (Goldberg,
2003). The transcription factor Rpn4p is a central regulator
of proteasomal abundance in the cell. In addition, Rpn4p
can associate with the 19S proteasomal cap, although the
purpose of this association is unknown. Rpn4p regulation
is complex and involves both ubiquitin-dependent and
ubiquitin-independent degradation by proteasomes. Moreover, the expression of RPN4 is induced under various
stress conditions that may require higher proteasome levels
(Hanna & Finley, 2007). Rpn4p and Rpn4p-dependent
processes have been implicated in As(III) tolerance; (1)
expression of proteasomal genes are strongly induced by
As(III), (2) this induction requires Rpn4p and (3) cells
lacking RPN4 are highly As(III) sensitive. Expression of
the RPN4 gene is also enhanced by As(III) and this induction requires the transcription factor Yap1p (Haugen et al.,
2004; Thorsen et al., 2007, 2009). These data suggest that
cells may increase the number of proteasomes and that
enhanced protein degradation is important for As(III)
tolerance. The latter is supported by the observation that
mutants defective in the ubiquitin–proteasome pathway
display severe As(III) sensitivity (Di & Tamás, 2007). Rpn4p
is also required for Cd tolerance (Thorsen et al., 2009) and
Cd-treated cells display increased protein degradation rates
(Medicherla & Goldberg, 2008). Finally, Cr triggers mRNA
mistranslation and protein aggregation, and proteasomal
activity is required for Cr tolerance (Holland et al., 2007).
Together, these data suggest that metal-treated cells accumulate damaged proteins and that cells need to remove
those proteins for optimal tolerance. Intriguingly, cells do
not only control proteasome abundance in response to
environmental stress, but proteasomes may also be subject
to other modes of regulation (Hanna & Finley, 2007). The
mammalian AIRAP protein has been shown to be specifically induced by As(III) and to associate with the 19S
proteasomal cap. The exact role of AIRAP in proteasome
function is unclear, but it may enhance proteasomal stability
and/or activity when bound to the 19S cap (Stanhill et al.,
2006). Whether Rpn4p would have a similar role is unknown.
Hog1p has a dual role in As(III) tolerance: As(III)
influx and cell cycle control
Eukaryotic cells respond to various stress conditions by
activating a family of serine/threonine kinases called
MAPKs. It is well known that the mammalian p38 MAPK
pathway plays a major role in As(III) tolerance by modulating gene expression via an AP-1 transcription factor (Elbirt
et al., 1998; Verma et al., 2002). Interestingly, As(III)
resistance of myeloma cell lines is associated with increased
activation of the p38 pathway (Wen et al., 2008). We have
shown that the MAPK Hog1p, a yeast homologue of p38, is
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R. Wysocki & M.J. Tamás
phosphorylated in response to As(III) and Sb(III), while
cells lacking Hog1p are highly sensitive to these metalloids
(Thorsen et al., 2006). In contrast to rapid, robust and
transient activation of Hog1p by osmotic stress (the main
trigger of Hog1p activation), As(III) and Sb(III) cause a
delayed and prolonged profile of Hog1p phoshorylation
(Sotelo & Rodriguez-Gabriel, 2006; Thorsen et al., 2006).
Importantly, Hog1p does not accumulate in the nucleus and
does not affect transcription in As(III)-exposed cells (Thorsen et al., 2006), suggesting that it mediates tolerance
through cytoplasmic targets. One such target is the aquglyceroporin Fps1p; Hog1p phosphorylates Fps1p and downregulates its transport activity, thereby reducing As(III)
influx. Deletion of HOG1 or expression of an Fps1p mutant
lacking the MAPK phosphorylation site at Thr231 results in
increased As(III) uptake and metalloid sensitivity (Thorsen
et al., 2006). Hog1p may also influence Fps1p indirectly by
modulating Rgc1p and Rgc2p. Rgc2p is phosphorylated in
the presence of As(III) and this phosphorylation is partially
Hog1p-dependent and epistasis analysis places RGC1 and
RGC2 between FPS1 and HOG1 (Beese et al., 2009). Hence,
Hog1p affects Fps1p activity in two ways: by directly
phosphorylating Fps1p on Thr231 (Thorsen et al., 2006)
and indirectly by downregulating the positive regulators of
Fps1p activity (Beese et al., 2009).
Hog1p also plays a crucial role under stress conditions by
controlling cell cycle progression. Under mild osmotic
stress, Hog1p contributes to cell cycle delay in all phases by
downregulating G1, S and G2 cyclins, by blocking the
degradation of Sic1p, which is an inhibitor of S-phase
cyclin-dependent kinase (Cdk)–cyclin complexes, by delaying the origin of replication firing and by stabilizing the
Swe1p kinase, which negatively regulates M-phase Cdk–
cyclin complexes (Escoté et al., 2004; Clotet et al., 2006;
Yaakov et al., 2009). The role of Hog1p in cell cycle
regulation is manifested by accelerated cell cycle progression
in the hog1D mutant exposed to osmotic stress or permanent cell cycle arrest during sustained activation of Hog1p.
Surprisingly, we found that the hog1D mutant is permanently
arrested in G1 in the presence of As(III) (Migdal et al., 2008).
This result suggests that Hog1p not only promotes cell cycle
arrest but also the recovery from stress-induced cell cycle
delay. Under prolonged As(III) exposure, G1-synchronized
hog1D cells exhibit prolonged stabilization of the CDKinhibitor Sic1p. Consistently, deletion of SIC1 in the hog1D
background suppresses persistent G1 delay. Hence, Hog1p
promotes recovery from As(III)-induced cell cycle arrest in
G1 by inducing the degradation of Sic1p and releasing the
activity of S-phase Cdk–cyclin complexes. However, cells
expressing a Sic1p mutant that cannot be phosphorylated by
Hog1p (Sic1p–Thr173Ala) do not display a cell cycle recovery
defect, suggesting that Hog1p indirectly regulates Sic1p
stability in the presence of As(III) (Migdal et al., 2008).
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Yeast metal tolerance
Hog1p is also phosphorylated upon Cd exposure with a
kinetics and magnitude that resembles its phosphorylation
by As(III) (Bilsland et al., 2004), but specific targets through
which Hog1p mediates Cd tolerance remain to be identified.
Hog1p homologues in C. albicans (CaHog1) and S. pombe
(Sty1/Spc1) are activated by As(III) and Cd (Smith et al.,
2004; Rodriguez-Gabriel & Russell, 2005). Moreover, deletion of Hog1p homologues in S. pombe and the fungal
pathogens C. albicans and Candida lusitaniae results in Cd
and As(III) sensitivity (Toone et al., 1998; Smith et al., 2004;
Rodriguez-Gabriel & Russell, 2005; Boisnard et al., 2008). In
contrast to S. cerevisiae, Cd treatment in C. albicans triggers
the osmostress-like response including rapid, but transient
phosphorylation, followed by nuclear accumulation of CaHog1 and a major reprogramming of transcription (Smith
et al., 2004; Enjalbert et al., 2006). Similarly, the response to
As(III) and Cd in S. pombe requires the transcription factor
Pap1, which is a downstream target of the MAPK Sty1/Spc1
(Toone et al., 1998; Rodriguez-Gabriel & Russell, 2005;
Kennedy et al., 2008). However, the mechanisms of metal
and metalloid tolerance induced by stress-activated MAP
kinases have not yet been revealed in these organisms.
Target of rapamycin (TOR) pathway and protein
kinase A (PKA): regulation of general stress
responses and ribosomal proteins
The TOR pathway is a major signalling network that
mediates temporal control of cell growth in eukaryotes.
The S. cerevisiae TOR pathway consists of two branches
formed by multiprotein complexes called TOR complex 1
(TORC1) and TOR complex 2 (TORC2). At the heart of
each complex is a serine/threonine protein kinase (Tor1p
and Tor2p), belonging to the phosphatidylinositol kinaserelated kinase (PIKK) family. Under favourable growth
conditions, TORC1 stimulates transcription, translation,
ribosome biogenesis and nutrient uptake, and at the same
time, inhibits protein degradation and autophagy as well as
the transcription of specific sets of genes involved in stress
and nutrient starvation responses. In contrast, TORC2 is
specifically required for cell cycle-dependent polarization of
the actin cytoskeleton, which is crucial for bud formation
(Wullschleger et al., 2006; Soulard et al., 2009). The downstream effectors of TORC1 in the activation of ribosomal
protein and ribosomal biogenesis gene transcription include
positive regulation of the Sch9p kinase, the forkhead transcription factor Fhl1p and the zinc finger-containing transcription factor Sfp1p (Marion et al., 2004; Martin et al.,
2004; Urban et al., 2007). In response to nitrogen starvation
and stress, TORC1 signalling is inhibited, leading to downregulation of protein synthesis, promotion of autophagy
and activation of several stress-responsive transcription
factors, including Msn2p and Msn4p (Wullschleger et al.,
FEMS Microbiol Rev 34 (2010) 925–951
2006). The TOR pathway acts in concert with another major
nutrient-responsive signal transduction pathway: the Ras/
cyclic AMP (cAMP)/PKA pathway. PKA consists of two
catalytic and two inhibitory subunits, and is activated by the
presence of cAMP to regulate cell growth and responses to
nutrients and stress. PKA controls a wide range of processes,
including transcription, energy metabolism and cell cycle
progression, and acts by modulating transcription factors,
enzymes and other regulatory kinases (Santangelo, 2006).
Recent findings implicate the TOR and PKA pathways in
metal tolerance by modulating the expression of genes
involved in the general stress response and protein synthesis
(Hosiner et al., 2009).
Genome-wide expression analyses, coupled to searches
for cis-regulatory elements in coregulated genes, implicated
Msn2p and Msn4p in the transcriptional response to As(III)
(Haugen et al., 2004; Thorsen et al., 2007; Kristiansson et al.,
2009). These proteins are partially redundant transcriptional activators with Cys2–His2 zinc finger DNA-binding
motifs, and they stimulate the expression of genes in
response to many stress conditions. Activation of these
‘general stress responsive’ transcription factors involves
translocation from the cytoplasm to the nucleus and binding to promoters containing the DNA sequence AAGGGG
(Martinez-Pastor et al., 1996; Schmitt & McEntee, 1996;
Görner et al., 1998; Gasch et al., 2000). The activity of
Msn2p/Msn4p is regulated by growth conditions and the
nutritional status of cells, and involves the PKA and TOR
pathways (Görner et al., 1998; Beck & Hall, 1999). In
response to As(III), Msn2p and Msn4p translocate to the
nucleus and stimulate the expression of target genes. Interestingly, deletion of both MSN2 and MSN4 results in
increased tolerance to As(III), whereas their overexpression
enhances sensitivity (Hosiner et al., 2009). Based on these
data, it was suggested that As(III) toxicity might be a
consequence of chronic activation of stress-responsive genes
via Msn2p/Msn4p (Hosiner et al., 2009). In line with this
notion, hyperactivation of these proteins has a negative
effect on cell growth (Durchschlag et al., 2004). Similarly,
overexpression of MSN2 sensitizes yeast cells to MeHg,
whereas MSN2 deletion slightly improves growth in the
presence of this agent (Hwang et al., 2005).
Genome-wide expression and computational analyses
also implicated Sfp1p, Fhl1p and Rap1p in controlling
As(III)-repressed genes (Haugen et al., 2004; Thorsen et al.,
2007; Kristiansson et al., 2009). These transcription factors
principally regulate the expression of genes involved in
ribosomal function. Sfp1p has been shown to be regulated
in response to nutrients and stress by the kinases TOR and
PKA (Marion et al., 2004; Hosiner et al., 2009). Sfp1p is
localized to the nucleus, where it binds to target-gene
promoters and promotes the expression of ribosomal proteins under optimal growth conditions. In response to
2010 Federation of European Microbiological Societies
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c
942
inhibition of TOR, changes in nutrient availability or stress
conditions, Sfp1p is released from DNA and leaves the
nucleus, which results in the downregulation of ribosomal
protein-gene expression (Marion et al., 2004). Accordingly,
As(III), but also Hg and Ni, inhibits TORC1 and reduces
Sfp1p-regulated gene expression. Moreover, As(III) treatment leads to Sfp1p dephosphorylation, dissociation from
chromatin and nuclear exit (Hosiner et al., 2009). Cells
lacking SFP1 cannot downregulate ribosomal protein-gene
expression in response to As(III). Curiously, while tor1D
cells are As(III) sensitive, SFP1 deletion results in enhanced
As(III) tolerance. Moreover, SFP1 overexpression sensitizes
cells to As(III) (Hosiner et al., 2009). Currently, it is not
completely clear how these apparently contradictory views
on the role of Sfp1p and TORC1 can be reconciled into a
coherent model.
Concluding remarks
We have learned a great deal in recent years about how
S. cerevisiae and other yeasts cope with toxic metals and
metalloids. Nevertheless, we still lack a molecular insight
into many aspects of metal biology. For example, genomewide phenotype screens and molecular work pinpointed
many proteins that protect cells from metal toxicity; yet, the
exact mechanism(s) by which these proteins mediate tolerance are largely elusive. We know very little about how
metals activate transcription factors and signalling proteins,
and how these proteins in turn activate their gene/protein
targets. We also know little about the post-translational
regulation of various transporters involved in metal tolerance. Importantly, how these proteins interact in space and
time as they orchestrate the cell’s response to metals on a
whole-cell or systems level has remained largely unexplored.
Other aspects of metal biology that have received limited
attention is whether cells respond differently under acute
and chronic exposure and whether colonies use strategies
that are distinct from those used by cells in (liquid) culture.
Finally, the insight into how metal tolerance systems have
evolved is only starting to emerge. We believe that
S. cerevisiae is an excellent model system for exploring these
fundamental aspects of metal biology; many large-scale
techniques and knock-out/overexpression collections are
available for identifying toxicity targets and tolerance systems, yeast is easily used as a heterologous host to study
proteins from other organisms and yeast is a front-runner
when it comes to the availability of (novel) systems biology
approaches to unveil fundamental mechanisms on a wholecell and/or a single-cell level. Because many mechanisms
involved in metal toxicity and detoxification appear to be
conserved in various eukaryotic organisms, such work in
yeast may prove of value for identifying similar mechanisms
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Published by Blackwell Publishing Ltd. All rights reserved
c
R. Wysocki & M.J. Tamás
in other organisms and have important implications for the
use of metals in medical therapy.
Acknowledgements
M.J.T. is supported by funds from the Swedish Research
Council and the Swedish Research Links programme, and
R.W. is supported by funds from the Polish Ministry of
Science and Higher Education. We thank Jean Labarre
(CEA, France), Jonas Warringer and Therese Jacobson
(University of Gothenburg, Sweden) for critically reading
the manuscript, and Donata Wawrzycka (University of
Wroclaw, Poland), Barry Rosen (Florida International University), Jean Labarre and Jonas Warringer for sharing
unpublished data. We wish to dedicate this paper to Beatrix
Tamás (1929–2009).
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